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PubMed1990

Cytolytic human lung lymphocytes: characterization of intragranular protease content and response to interleukin-2.

Cytolytic lymphocytes play an important role in defense against viral and neoplastic disease. Integral to the function of these cells is the content of lysosomal granules. Recent attention has focused on a family of proteases present in the granules of natural killer (NK) cells, interleukin-2 (IL-2)-activated NK cells (LAK cells), and cytotoxic T lymphocytes (CTL). In the current investigation, lymphocytes were obtained from human lung parenchyma and peripheral blood. Following activation with IL-2, both groups of lymphocytes exhibited comparable cytolytic activity against K562 targets. Lysosomal granules obtained from these cells contained two serine proteases with molecular weights of 30 and 28 kD. These proteases were capable of hydrolyzing benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT-ester), a substrate of cytolytic lymphocyte proteases. When compared to blood, unactivated lung lymphocytes contained significantly higher levels of protease content. Although IL-2 produced a significant increase in blood lymphocyte protease content, no change in lung lymphocyte granule protease activity was observed. We conclude that cytolytic lung lymphocytes contain high levels of lysosomal granule protease but differ from blood lymphocytes in the ability to increase protease content following activation with IL-2. The high level of protease content in cytolytic lung lymphocytes suggests that these cells could produce local tissue injury during the release of lysosomal granules.

Cytoplasmic Granules↗
PubMed2006

Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis.

BACKGROUND: Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. RESULTS: Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. CONCLUSION: Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae.

Acanthamoeba↗
PubMed2006

Protease gene families in Populus and Arabidopsis.

BACKGROUND: Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. RESULTS: We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. CONCLUSION: Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

Arabidopsis↗
PubMed1997

Profiles of insulin-like growth factor binding proteins and the protease activity in the maternal circulation and its local regulation between placenta and decidua.

Insulin-like growth factors (IGF) and their specific binding proteins (IGFBPs) are believed to be important regulators of fetal growth. IGFBP protease which proteolyzes IGFBPs and changes the biochemical properties of IGFBPs is also presumed to be involved in fetal growth. The aim of this study is to elucidate the physiological significance of IGFBP protease in fetal growth and regulators of protease in placenta and decidua. The intact IGFBP-3 was proteolyzed into fragments when pregnant serum was incubated with 125I-IGFBP-3. IGFBP-3 protease activity showed a marked increase at 5 weeks of gestation and reached a plateau in maternal circulation at 15 weeks of gestation. These changes in protease activity correlated with the profiles of IGFBPs in the maternal circulation analyzed by Western ligand blot, where the IGFBP-1 is only the dominant IGFBP. The intact IGFBP-3 was proteolyzed when culture media of decidual cells were incubated with 125I-IGFBP-3, but was not proteolyzed when culture media of trophoblast cells were incubated with 125I-IGFBP-3. Decidual protease activity was slightly increased by IGF-I and completely inhibited by progesterone. The protease activity was more increased in the mothers with growth retarded infant than in those in the mothers with normal growth infants, suggesting that the protease activity is elevated in compensation for the impaired fetal growth. These results suggest that increased protease activity in maternal blood may be involved in the fetal growth indirectly by reducing the binding activity of IGFBP-3 to IGF-I, and that protease activity in maternal blood may be derived from decidua that is regulated by placental hormones.

Adult↗
PubMed1993

Yeast extracellular proteases.

Many species of yeast secrete significant amounts of protease(s). In this article, results of numerous surveys of yeast extracellular protease production have been compiled and inconsistencies in the data and limitations of the methodology have been examined. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisiae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized. Biotechnological applications and the medical relevance of yeast extracellular proteases are covered. Yeast extracellular proteases have potential in beer and wine stabilization, and they probably contribute to pathogenicity of Candida spp. Yeast extracellular protease genes also provide secretion and processing signals for yeast expression systems designed for secretion of heterologous proteins. Coverage of the secretion of foreign proteases such as prochymosin, urokinase, and tissue plasminogen activator by yeast in included.

Amino Acid Sequence↗
PubMed1975

Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.

Animals↗
PubMed1997

Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media.

When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes. The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2. A metallo protease component (SV3) and an azocaseinase component (SV4) were also present. Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8. Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4. This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit. The method chosen for initial downstream processing influenced both the yield and type of protease purified. When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered. The medium used for the growth of Strep. thermovulgaris also appeared to affect the type of protease produced. A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.

Chromatography, Ion Exchange↗
PubMed2003

Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis.

Many serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C, TMPRSS2, hepsin, matriptase/MT-SP1, dipeptidylpeptidase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis.

Acrosin↗
PubMed1992

Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580.

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.

Amino Acid Sequence↗
PubMed1990

Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin.

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.

Animals↗
PubMed1988

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase.

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.

Cations, Divalent↗
PubMed1988

Proteases in cyst fluid from human gross cyst breast disease.

Cyst fluid from women with gross cystic breast disease was found to contain protease activity when assayed against [14C]albumin. At least six different proteases were detected when the fluid was fractionated by a combination of S-300 Sephacel, hydroxylapatite, and DEAE-Sephacel chromatographic techniques. The distribution of the proteases appeared to be related to the ionic composition of the fluids. A major protease component, found in both high Na and high K fluids, was isolated. It showed chymotryptic cleavage characteristics against the beta-chain of insulin. It was partially inhibited by alpha 2-macroglobulin, N-tosyl-L-phenylalanine chloromethyl ketone, and benzamidine but not by leupeptin, pepstatin, N-tosyl-L-lysine chloromethyl ketone, or alpha 1-protease inhibitor. The protease has an apparent molecular weight of 110,000 with Mr 24,000 subunits. This protease may be identical or closely associated with Haagensen's GCDFP-24 progesterone binding protein which was isolated in a similar manner. An imbalance between protease and protease inhibitors in cyst fluid may account for gross cyst formation and may be involved in the tumorigenic process. The accumulation of poorly diffusible peptide fragments, as a result of protease activity, would increase the oncotic pressure leading to enlargement of the cyst cavity as water enters to reestablish osmotic equilibrium.

Female↗
PubMed1984

Multiple forms of Ca-activated protease from rat brain and muscle.

Three Ca-dependent proteases have been identified in rat brain and skeletal muscle using ion exchange, gel filtration, and substrate affinity chromatography. A high degree of homology exists among three enzymes from different sources. Both the high molecular weight protease (154,000) and lower molecular weight protease (96,000) show high affinity for calcium while the third protease (76,000) had low affinity for calcium. Transformation among the three enzymes was calcium-induced and the process was unidirectional, generating a lower molecular weight form with decreased affinity for calcium. The protease with low affinity for calcium was susceptible to calcium-induced inactivation by autocatalysis. Immunologically the three proteases were equivalent, if not identical, and the brain and muscle proteases cross-react. All three proteases degraded neurofilament proteins; however, the protease with low affinity for calcium had 3 to 6 times higher specific activity. It is suggested that the high molecular weight enzyme (154,000) may be the native form of the Ca-dependent protease present in vivo.

Animals↗
PubMed1983

Purification and characterization of an acid protease and vasopeptide kinins from Murphy-Sturm lymphosarcoma.

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was isolated, purified and characterized. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. The purification was carried out in seven steps involving homogenization of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation to 60% saturation, DEAE-Sephadex batch treatment, chromatography on a QAE-Sephadex column, gel filtration on Sephadex G-200 and finally chromatography on CM-32 cellulose. The purification scheme resulted in a 640-fold purification and a homogeneous preparation as confirmed by disc gel electrophoresis and Ouchterlony immunodifussion. Sephadex G-200 elution profiles indicated two different protease fractions containing protease activity which showed a single band with identical relative mobility on polyacrylamide gel electrophoresis in the presence and absence of SDS, and also showed immunological cross-reactivity against anti-acid protease antiserum raised in the rabbit against one of the fractions. The acid protease is presumed to be a single protease which has a tendency to aggregate. An apparent molecular weight of 39,500 was estimated by gel filtration on Sephadex G-200, and 41,000 by polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis showed that the acid protease was comprised of a major band with a molecular weight of 4,000 and two fainter bands with molecular weights of 27,000 and 12,000. The purified enzyme showed three major isozymic forms (alpha, beta, gamma) which had isoelectric points (pI) of 5.2, 5.5 and 5.8 respectively, and had near identical amino acid compositions. The carbohydrate moiety contained 2 mol of N-acetylglucosamine and 8 mol of mannose per mol enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0. The protease activity was very stable above pH 3.4. The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents per mg substrate after 2 hr incubation at 37 degrees. The vasopeptide was purified to electrophoretic homogeneity by gel filtration on Sephadex G-50 and chromatography on a column of CM-Sephadex. The amino acid composition of vasopeptide was Ser2, Gly1, Pro4, Ile1, Leu1, Phe2, Arg2.

Amino Acids↗
PubMed1995

Developmentally regulated secretion of cathepsin L-like cysteine proteases by Haemonchus contortus.

Cysteine protease activity was present in media collected after 24 hr in vitro culture of adult Haemonchus contortus. The released cysteine protease hydrolyzed the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-substituted synthetic peptides Z-phe-arg-AFC and Z-ala-arg-arg-AFC, but not Z-arg-arg-AFC or Z-arg-AFC, characterizing this activity as cathepsin L-like. Within the parasite, cysteine protease activity was highest in extracts of intestinal tissue. Secreted cysteine protease inhibited the clotting of sheep blood and hydrolyzed hemoglobin, fibrinogen, collagen, and IgG; the IgG hydrolysis site was within the hinge region. Four proteases with M(r) values of 30, 34, 37, and 41 kDa were identified with biotinylated-phenylalanine-arginine-fluoromethyl ketone, a specific probe that binds to active cysteine proteases. Adult parasites cultivated in the presence of 0.1 mM levamisole released 50% less protease activity compared to control cultures; in the presence of rafoxanide (0.1 mM), protease was not detected. Cathepsin L-like cysteine protease activity was released also by L4, but not the L3 larval stage. The active and developmentally regulated release of cysteine proteases by H. contortus may have a critical function in worm nutrition, immune evasion, or both.

Amino Acid Sequence↗
PubMed1996

Design and synthesis of substrate-based peptidomimetic human immunodeficiency virus protease inhibitors containing the hydroxymethylcarbonyl isostere.

The human immunodeficiency (HIV) codes for an aspartic protease known to be essential for retroviral maturation and replication. The HIV protease can recognize Phe-Pro and Tyr-Pro sequences as the virus-specific cleavage site. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treatment of acquired immunodeficiency syndrome (AIDS). HIV protease is formed from two identical 99 amino acid peptides. We replaced the two Cys residues by L-Ala to synthesize [Ala67,95]-HIV-1 protease by the solid phase method and then prepared [Tyr6,42, Nle36,46, (NHCH2COSCH2CO)51-52, Ala67,95] HIV-1 protease (NY-5 isolate) using the thioester chemical ligation method. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing an unnatural amino acid, (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) with a hydroxymethylcarbonyl (HMC) isostere. Among them, the conformationally constrained tripeptide kynostatin (KNI)-272 (iQoa-Mta-Apns-Thz-NHBut) was a highly selective and superpotent HIV protease inhibitor (Ki = 0.0055 nM). KNI-272 exhibited potent antiviral activities against both AZT-sensitive and -insensitive clinical HIV-1 isolates as well as HIV-2 with low cytotoxicity. After i.d. administration, bioavailability of KNI-272 was 42.3% in rats. Also, KNI-272 exhibited in vivo anti-HIV activities in human PBMC-SCID mice. The x-ray crystallography and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of HIV protease active site in the essentially same hydrogen-bonding mode as the transition state. This result implies that the HMC isostere is an ideal transition-state mimic and contributes to the high activity of KNI-272.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Reactivity of cysteine residues in the protease from human immunodeficiency virus: identification of a surface-exposed region which affects enzyme function.

The protease encoded by the human immunodeficiency virus (HIV) is essential for the processing of viral polyproteins encoded by the gag and pol genes into mature viral proteins. The 99-residue protease from HIV-1 contains two cysteine residues (Cys-67 and Cys-95), both of which are usually conserved in viruses isolated from patients. Despite this conservation, neither residue is required for enzymatic activity. Certain site-specific cysteine mutants of HIV-1 protease are catalytically active, and the protease from HIV-2 lacks both cysteines. Copper is a potent inhibitor of HIV-1 protease, but not of mutants lacking cysteine (A. R. Karlström and R. L. Levine, 1991, Proc. Natl. Acad. Sci. USA 88, 5552-5556). The addition of copper to the protease at pH 5.5 induced aggregation of the protein, providing a possible basis for the inhibitory action of copper. However, addition of both copper and dithiothreitol still led to inhibition of activity but did not cause aggregation. These findings led to a study of the reactivity of the cysteine residues to 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent), a sulfhydryl compound which reacts with the ionized form of cysteine residues. At pH 6.2 in 6 M guanidine, no derivatization of cysteine residues occurred, consistent with the typical pKa of cysteine expected for the denatured protein. However, in the same buffer without guanidine, the native protease reacted rapidly with concomitant loss of proteolytic activity. Peptic mapping demonstrated that both Cys-67 and Cys-95 were derivatized. A catalytically active fusion protein of protease with protein A domains was then studied with the expectation that access to Cys-95 would be hindered. This was confirmed, with only Cys-67 reacting rapidly with Ellman's reagent. Enzymatic activity was again lost, indicating that derivatization of the surface-accessible Cys-67 was sufficient to inactivate the enzyme. The reactivity and accessibility of these residues suggest an interesting approach for the development of protease inhibitors which are not directed to the substrate-binding site.

Copper↗
PubMed1995

Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+.

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for processing viral polyproteins which contain the enzymes and structural proteins required for the infectious virus. It was previously found that cupric chloride, in the presence of dithiothreitol or ascorbic acid, could inhibit the HIV-1 protease. It was suggested that a Cu1+ chelate was the moiety responsible for inhibition of the protease. This hypothesis has now been investigated directly by utilizing the stable Cu1+ chelate, bathocuproine disulfonic acid Cu1+ (BCDS-Cu1+). BCDS-Cu1+ inhibited the HIV-1 wild type protease as well as a mutant HIV-1 protease lacking cysteines. BCDS-Cu1+ was a competitive inhibitor of the mutant HIV-1 protease with an apparent Ki of 1 microM. Replication of HIV-1 in human lymphocytes and the cytotoxic effect of HIV-1 in CEM cells was inhibited by micromolar BCDS-Cu1+. Inhibition of the protease and of HIV replication by BCDS-Cu1+ was dependent on the presence of Cu1+ as BCDS alone was ineffective. EDTA blocked the inhibition of the protease by Cu1+ but was unable to block inhibition of the protease by BCDS-Cu1+, indicating that the Cu1+ complex was the inhibitory agent. The apparent IC50 for BCDS-Cu1+ on the inhibition of replication by primary isolates of HIV-1 was 5 microM. However, BCDS-Cu1+ did not affect polyprotein processing in an H9 cell line chronically infected with HIV-1, indicating that BCDS-Cu1+ acts by yet another mechanism to block HIV infection. Other possible targets for BCDS-Cu1+ include inhibition of viral adsorption and/or inhibition of the HIV-1 integrase.

Antiviral Agents↗